Peptide compounds for suppressing inflammation

ABSTRACT

Provided herein are peptides that exhibit ApoE biological activity, as well as compositions and pharmaceutical formulations that include the peptides. The peptides, compositions, and methods disclosed herein have broad applications as they can be used to treat a broad spectrum of injury, diseases, disorders, and clinical indications.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/840,695, filed Jun. 28, 2013, and:

This application is a continuation of U.S. application Ser. No.16/299,793, filed Mar. 12, 2019, which is a continuation of U.S.application Ser. No. 15/054,563, filed Feb. 26, 2016, now U.S. Pat. No.10,280,210, which is a continuation of U.S. application Ser. No.14/278,643, filed May 15, 2014, now U.S. Pat. No. 9,303,063, whichclaims the benefit of U.S. Provisional Application Ser. No. 61/840,695,filed Jun. 28, 2013, and is a continuation-in-part of commonly ownedU.S. application Ser. No. 13/981,238, filed Oct. 15, 2013, now U.S. Pat.No. 9,018,169, which is a filing under 35 U.S.C. section 371 ofInternational Application No. PCT/US2012/029392, filed Mar. 16, 2013,and published Sep. 27, 2012, which in turn claims priority from U.S.Provisional Application Ser. No. 61/454,342, filed Mar. 18, 2011, thedisclosures of each of which are incorporated by reference herein intheir entirety.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 1175-2TSIPCT2_ST25.txt, 12,725 bytes in size, generatedon Mar. 11, 2019 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

FIELD

The disclosure relates to peptides, methods, and compositions forreducing or suppressing inflammation, reducing or suppressingneuroinflammation, and treating neurological conditions.

BACKGROUND

Apolipoprotein E (“ApoE”) is a 299 amino acid (34 kDa) glycoprotein,produced primarily in the liver and brain that exhibits multiplebiological functions. First recognized for its role in cholesteroltransport and metabolism, ApoE is present in very-low-densitylipoprotein (VLDL) and high-density lipoprotein (HDL) complexes, andApoE can bind the low-density lipoprotein (LDL) receptor, theLDL-receptor-related protein (LRP), and the VLDL receptor. Weisgraber,(1994) Adv. Protein Chem. 45:249-302. ApoE is also known to haveimmunomodulatory properties, Laskowitz, et al., (2001) Exp. Neurol.167:74-85, and to play a role in neurological disease and brain injuryresponse, Laskowitz and Vitek, (2007) Pharmacogenomics 8:959-69.

The tertiary structure of ApoE includes an amino-terminal region with afour-α-helix motif that includes a receptor-binding domain and acarboxy-terminal region that is largely responsible for lipid binding.The receptor-binding region of ApoE has been mapped to a helical domainat residues 130-150 of the mature full-length protein, and this regionof ApoE governs its ability to suppress glial activation and CNSinflammation.

SUMMARY

In an aspect, the disclosure provides a peptide of 5, 6, 7, 8, or 9amino acid residues that comprises Formula I:

X1-X2-X3-X4-X5  (SEQ ID NO:1)

or a salt thereof, wherein X1 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain; X2 is selected from an amino acid having a hydrophobic sidechain, an amino acid having a positively charged side chain, or an aminoacid having a polar uncharged side chain; X3 is selected from an aminoacid having a positively charged side chain; X4 is selected from anamino acid having a positively charged side chain; and X5 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain. Some embodiments of this aspectprovide for a peptide of Formula II:

X1-X2-X3-X4-X5-X6-X7-X8-X9  (SEQ ID NO:17);

wherein X1, X2, X3, X4, and X5 are as noted above, and each of X6, X7,X8, and X9 are independently selected from any amino acid, and areoptionally absent.

In an aspect, the disclosure provides a peptide of Formula I:

X1-X2-X3-X4-X5  (SEQ ID NO:1)

or a salt thereof, wherein X1 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain; X2 is selected from an amino acid having a hydrophobic sidechain, an amino acid having a positively charged side chain, or an aminoacid having a polar uncharged side chain; X3 is selected from an aminoacid having a positively charged side chain; X4 is selected from anamino acid having a positively charged side chain; and X5 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain.

In embodiments of this aspect, the disclosure provides a peptide, or asalt thereof, according to SEQ ID NO:1, wherein X1 is V or R; X2 is S,A, or H; X3 is K or R; X4 is K or R; and X5 is R, L, or K. In someembodiments of this aspect, the disclosure provides a peptide, or saltthereof, comprises VSRKR (SEQ ID NO:2), VSKRR (SEQ ID NO:3), VSRRR (SEQID NO:4), VARKL (SEQ ID NO:5), RHKKL (SEQ ID NO:6), RARRL (SEQ ID NO:7),RSKKL (SEQ ID NO:8), RHKRR (SEQ ID NO:9), VARRL (SEQ ID NO:10), VARRK(SEQ ID NO:11), or RSKRR (SEQ ID NO: 12).

In an aspect, the disclosure provides for compositions comprising thepeptide of Formula I, and a carrier, diluent, vehicle, or adjuvant.Embodiments of this aspect provide for a composition comprising apeptide, or a salt thereof, according to SEQ ID NO:1, wherein X1 is V orR; X2 is S, A, or H; X3 is K or R; X4 is K or R; and X5 is R, L, or K.In some embodiments of this aspect, the disclosure provides acomposition comprising a peptide, or salt thereof, of VSRKR (SEQ IDNO:2), VSKRR (SEQ ID NO:3), VSRRR (SEQ ID NO:4), VARKL (SEQ ID NO:5),RHKKL (SEQ ID NO:6), RARRL (SEQ ID NO:7), RSKKL (SEQ ID NO:8), RHKRR(SEQ ID NO:9), VARRL (SEQ ID NO:10), VARRK (SEQ ID NO:11), or RSKRR (SEQID NO: 12).

In an aspect, the disclosure provides a method of reducing inflammationin a subject in need thereof, the method comprising administering to thesubject an effective amount of a peptide of Formula I:

X1-X2-X3-X4-X5  (SEQ ID NO:1)

or a salt thereof, wherein X1 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain; X2 is selected from an amino acid having a hydrophobic sidechain, an amino acid having a positively charged side chain, or an aminoacid having a polar uncharged side chain; X3 is selected from an aminoacid having a positively charged side chain; X4 is selected from anamino acid having a positively charged side chain; and X5 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain.

In embodiments of this aspect, the method comprises a peptide or a saltthereof according to SEQ ID NO:1, wherein X1 is V or R; X2 is S, A, orH; X3 is K or R; X4 is K or R; and X5 is R, L, or K. In some embodimentsof this aspect, the method provides a peptide, or salt thereof,comprising VSRKR (SEQ ID NO:2), VSKRR (SEQ ID NO:3), VSRRR (SEQ IDNO:4), VARKL (SEQ ID NO:5), RHKKL (SEQ ID NO:6), RARRL (SEQ ID NO:7),RSKKL (SEQ ID NO:8), RHKRR (SEQ ID NO:9), VARRL (SEQ ID NO:10), VARRK(SEQ ID NO:11), or RSKRR (SEQ ID NO:12).

In an aspect, the disclosure provides a method of treating aneurological condition in a subject in need thereof, the methodcomprising administering to the subject an effective amount of a peptideof Formula I:

X1-X2-X3-X4-X5  (SEQ ID NO:1)

or a salt thereof, wherein X1 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain; X2 is selected from an amino acid having a hydrophobic sidechain, an amino acid having a positively charged side chain, or an aminoacid having a polar uncharged side chain; X3 is selected from an aminoacid having a positively charged side chain; X4 is selected from anamino acid having a positively charged side chain; and X5 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain.

In embodiments of this aspect, the method comprises a peptide or a saltthereof according to SEQ ID NO:1, wherein X1 is V or R; X2 is S, A, orH; X3 is K or R; X4 is K or R; and X5 is R, L, or K. In some embodimentsof this aspect, the method provides a peptide, or salt thereof,comprising VSRKR (SEQ ID NO:2), VSKRR (SEQ ID NO:3), VSRRR (SEQ IDNO:4), VARKL (SEQ ID NO:5), RHKKL (SEQ ID NO:6), RARRL (SEQ ID NO:7),RSKKL (SEQ ID NO:8), RHKRR (SEQ ID NO:9), VARRL (SEQ ID NO:10), VARRK(SEQ ID NO:11), or RSKRR (SEQ ID NO:12). In some embodiments the methodcomprises treating a neurological condition selected from at least oneof traumatic CNS injury, subarachnoid hemorrhage, intracranialhemorrhage, stroke, experimental allergic encephalomyelitis, multiplesclerosis, neuroinflammation, chronic neurological disease, ALS,dementia, neuropathy, epilepsy, Parkinson's disease, and Alzheimer'sdisease.

In other aspects the disclosure provides a medicament comprising atleast one peptide of formula I, methods for the preparation of themedicament, and a method comprising administration of the medicament asdescribed herein.

In various embodiments of the aspects discussed above, the disclosurerelates to and provides for peptides that consist essentially of, orconsist of, the recited sequences and formulae.

The disclosure provides for additional aspects and embodiments that willbe apparent to one of ordinary skill in the art in light of the drawingsand detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the inhibition of microglial TNF-α secretion by apeptide. Cultured BV-2 murine microglial cells were incubated witheither the indicated concentrations of peptide (SEQ ID NO:3) or anegative control peptide (SEQ ID NO:13) and stimulated withlipopolysaccharide (LPS) (100 ng/mL) for six hours. After six hours,supernatants were harvested and the concentration of secreted TNF-α wasmeasured by ELISA (** p<0.01; ANOVA).

FIG. 2 depicts improved vestibulomotor function by mice treated withpeptide following traumatic brain injury. Vestibulomotor function wasassessed in terms of rotorod latency. Baseline rotorod latency wasassessed before (Day 0) and each day following traumatic brain injuryinduced by a controlled pneumatic impact against the intact skull. Attwo hours and six hours after traumatic brain injury, mice receivedeither peptide (SEQ ID NO:3; 0.05 mg/kg or 0.2 mg/kg) or saline vehicleby intravenous tail vein injection. Animals treated with peptideexhibited improved vestibulomotor performance relative to treatment withvehicle, as reflected by increased rotorod latency, throughout thetesting period (* p<0.05; ANOVA).

FIG. 3 depicts improved vestibulomotor function by mice treated withpeptide following traumatic brain injury. Vestibulomotor function wasassessed in terms of rotorod latency. Baseline rotorod latency wasassessed before (Day 0) and each day following traumatic brain injuryinduced by a controlled pneumatic impact against the intact skull. Attwo hours and six hours after traumatic brain injury, mice receivedeither peptide (SEQ ID NO:4; 0.05 mg/kg) or saline vehicle byintravenous tail vein injection. Animals treated with peptide exhibitedimproved vestibulomotor performance, as reflected by increased rotorodlatency, throughout the testing period (* p<0.05; repeated measuresANOVA).

FIG. 4 depicts improved neurocognitive outcomes by mice treated withpeptide following traumatic brain injury induced by a controlledpneumatic impact against the skull. At two hours and six hours aftertraumatic brain injury, mice received either peptide (SEQ ID NO:4, 0.05mg/kg or 0.2 mg/kg) or saline vehicle by intravenous tail veininjection. Neurocognitive performance was assessed in terms of Morriswater maze latency. Water maze latency was assessed by submergedplatform testing starting on day 28 after traumatic brain injury, andperformance was further evaluated on successive days 29, 30, and 31post-injury. Animals treated with peptide exhibited improvedneurocognitive outcomes, as reflected by decreased water maze latency,throughout the testing period (* p<0.05; ANOVA).

FIG. 5 depicts improved vestibulomotor function by mice treated withpeptide following intracerebral hemorrhage. Vestibulomotor function wasassessed in terms of rotorod latency. Baseline rotorod latency wasassessed before (Day 0) and each day following intracerebral hemorrhageinduced by stereotactic collagenase injection. At two hours and sixhours after collagenase injection, mice received either peptide (SEQ IDNO:4; 0.05 mg/kg) or saline vehicle by intravenous tail vein injection.Animals treated with peptide exhibited improved vestibulomotorperformance, as reflected by increased rotorod latency, throughout thetesting period (* p<0.05; repeated measures ANOVA).

FIG. 6 depicts the percent suppression of LDH release after NMDAexposure in cultures treated with ApoE mimetic peptide (SEQ ID NO: 4).

FIG. 7, Panels A and B depict sections of the shock tube blast modelapparatus.

FIG. 8 depicts the efficacy of ApoE mimetic peptide (SEQ ID NO:4) onneurocognitive performance of mice with blast injury (p=0.7 on learningtrend differences) in terms of Morris water maze latency.

FIG. 9 depicts the average and individual amounts of VSRRR (SEQ ID NO:4)peptide in plasma of mice over time after a single dose of 0.8 mg/kg.

FIG. 10 depicts the CNS penetration of VSRRR (SEQ ID NO:4) peptide inbrain tissue from mice over time.

FIG. 11, Panels A and B depict the inhibition of microglial TNF-αsecretion by VR-55 (SEQ ID NO: 4), VL-5-1 (SEQ ID NO: 5) and VL-5-3 (SEQID NO: 10).

FIG. 12, Panels A and B depict the vestibulomotor function of micetreated with peptide following modified middle cerebral arteryocclusion.

DETAILED DESCRIPTION

It will be understood that the various aspects and embodiments describedherein are merely intended to provide illustration and do not serve tolimit the scope of the claims.

Articles “a” and “an” are used herein to refer to one or to more thanone (i.e. at least one) of the grammatical object of the article. By wayof example, “an element” means at least one element and can include morethan one element. Unless otherwise defined, all technical terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs.

In a general sense the disclosure relates to peptides, includingisolated and/or synthetic peptides that can exhibit ApoE activity (alsoreferred to herein as “ApoE mimetic activity”). ApoE, as used herein,relates to any of the various isoforms (e.g., alleles) of the humanapolipoprotein-E protein encoded by an APOE gene. Non-limiting examplesof ApoE include ApoE3 (SEQ ID NO:14), ApoE2 (SEQ ID NO:15), and ApoE4(SEQ ID NO:16). ApoE activity includes any functional biologicalactivity, or combination of biological activities, that is associatedwith the ApoE protein, either in vitro or in vivo. ApoE activity canrelate to, for example, any one or combination of cholesterolmetabolism, binding to physiological ApoE receptor proteins;neuroprotective activity, antioxidant activity, anti-excitoticactivities, modulation of glial cell activity, inflammation, modulationof neuroinflammation, and the like. Recent studies demonstrate thatpeptides having apoE mimetic activity can beneficial effect in a numberof animal models including, for example, Alzheimer's disease (Laskowitzet al., (2010) J Neurotrauma 27:1983-1995); multiple sclerosis (Li etal., JPET, 2006); subarachnoid hemorrhage (Gao et al; 2006 Mesis et al.,2006); stroke (Tukhovskaya, J Neurosci Res, 2006), and neuropathy (Li etal., JPET, 2010). Thus, the peptides, compositions, and methodsdisclosed herein have broad applications as they can be used to treat aspectrum of diseases, disorders, and clinical indications associatedwith ApoE.

ApoE is a known ligand for receptors including scavenger receptors suchas LDL receptor, VLDL receptor, LRP/α2M receptor, ER-2 receptor, LR8receptor, ApoE receptor 2 (apoER2), and megalin/gp330 (collectively“ApoE receptors”). One region of ApoE known to participate inreceptor-binding interactions is an α-helical domain that lies betweenresidues 130-150 of the native ApoE polypeptide (SEQ ID NO:14). ActiveApoE fragments that include this helical domain have shown ApoE mimeticactivity (see U.S. Pat. Nos. 7,319,092 and 7,205,280, herebyincorporated by reference in their entireties). These peptides retainthe native ApoE primary amino acid sequence in the receptor bindinghelical domain and preserve the native α-helical secondary structure(Laskowitz, et al. (2001) Exp. Neurol. 167:74-85). The activity of thesepeptides has been shown to be dependent on the retention of the nativeα-helical secondary structure (Laskowitz, et al. (2006) Acta Neurol.Scand. 114 (Supp. 185):15-20). As described in more detail below, it hasbeen surprisingly found that small peptides (e.g., 1, 2, 3, 4, 5, 6, 7,8, or 9 amino acid residues in length) that have no primary sequenceidentity to ApoE are effective to modulate, induce, and/or mimic ApoEbiological activity and can be used in the treatment of variousdiseases, disorders, or conditions that involve ApoE biologicalfunction.

A “peptide” as used herein refers to a compound that comprises at leastsingle amino acid residue, or derivative thereof, or a compound thatcomprises at least one amino acid mimetic. Amino acids are well known inthe art and include, for example, isoleucine, leucine, alanine,asparagine, glutamine, lysine, aspartic acid, glutamic acid, methionine,cysteine, phenylalanine, threonine, tryptophan, glycine, valine,proline, serine, tyrosine, arginine, histidine, norleucine, ornithine,taurine, selenocysteine, selenomethionine, lanthionine,2-aminoisobutyric acid, dehydroalanine, hypusine, citrulline,3-aminopropanoic acid, gamma-aminobutryic acid, nitroarginine,N-methylated leucine, homoarginine, dimethyl arginine, acetyl lysine,azalysine, pyrrolysine, and the like. An “amino acid side chain” refersto the various organic substituent groups that differentiate one aminoacid from another. An amino acid having a hydrophobic side chainincludes the non-limiting examples of alanine (A), isoleucine (I),leucine (L), methionine (M), phenylalanine (F), tryptophan (W), tyrosine(Y), and valine (V). An amino acid having a positively charged sidechain, under typical physiological conditions, includes the non-limitingexamples of arginine (R), histidine (H), and lysine (K). An amino acidhaving a negatively charged side chain, under typical physiologicalconditions, includes the non-limiting examples of aspartic acid (D) andglutamic acid (E). An amino acid having a polar uncharged side chainincludes the non-limiting examples of serine (S), threonine (T),asparagine (N), and glutamine (Q). Given these non-limiting examples,one of skill in the art will appreciate and be able to determine thecharacteristics of other amino acid side chains (e.g., as hydrophobic,positively/negatively charged, polar uncharged, and the like) that arenot explicitly exemplified above. A “derivative” of an amino acid sidechain refers to an amino acid side chain that has been modifiedstructurally (e.g., through chemical reaction to form new species,covalent linkage to another molecule, and the like). Some embodimentsprovide for a peptide comprising modifications including, but notlimited to, glycosylation, side chain oxidation, acetylation, amidation,or phosphorylation, as long as the modification does not destroy thebiological activity of the peptides as herein described. For example, insome embodiments, a peptide may be modified by N-terminal acetylationand/or C-terminal amidation.

An “amino acid mimetic” as used herein is meant to encompasspeptidomimetics, peptoids (poly-N-substituted glycines) and β-peptides(i.e., peptides that comprise one or more amino acids residues havingthe amino group attached at the β-carbon rather than the α-carbon).Suitably, the amino acid mimetic comprises an altered chemical structurethat is designed to adjust molecular properties favorably (e.g.,stability, activity, reduced immunogenic response, solubility, etc.).Typically, the altered chemical structure is thought to not occur innature (e.g., incorporating modified backbones, non-natural amino acids,etc.). Thus, non-limiting examples of amino acid mimetic includeD-peptides, retro-peptides, retro-inverso peptides, β-peptides,peptoids, and compounds that include one or more D-amino acids,poly-N-substituted glycine, or R-amino acid, or any combination thereof.

Typically, a peptide comprises a sequence of at least 3 amino acids(amino acid residues) or amino acid mimetics. Embodiments of thedisclosure relate to small peptides of at least 1, 2, 3, 4, 5, 6, 7, 8,or 9 amino acid residues, mimetics, or combinations thereof. Someembodiments described herein provide for peptides of fewer than 9, 8, 7,6, 5, 4, 3, or 2 amino acid residues and/or mimetics. Some embodimentsrelate to peptides that are 5 amino acids in length. The peptidesdescribed herein can be provided in a charged form, typically with a netpositive charge, and can be generated and used as salts (e.g., alkalimetal salts, basic or acidic addition salts). The selection andformation of such salts are within the ability of one skilled in theart. See, e.g., Remington: The Science and Practice of Pharmacy, 21^(st)ed., Lippincott Williams & Wilkins, A Wolters Kluwer Company,Philadelphia, Pa. (2005).

Embodiments of the disclosure provide synthetic peptides with ApoEmimetic activity. Though they may exhibit ApoE mimetic activity, thedisclosed peptides do not share primary protein sequence identity withthe ApoE polypeptide (SEQ ID NO: 13). In other words, the disclosedpeptide sequences do not appear in the primary amino acid sequence of anApoE polypeptide, nor do they exhibit α-helical secondary structureanalogous to the native ApoE receptor-binding domain. In an embodiment,the synthetic peptides are optionally isolated and/or purified to asingle active species.

In an aspect of the disclosure, the peptide comprises Formula I:

X1-X2-X3-X4-X5  (SEQ ID NO:1)

or a salt thereof, wherein X1 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain; X2 is selected from an amino acid having a hydrophobic sidechain, an amino acid having a positively charged side chain, or an aminoacid having a polar uncharged side chain; X3 is selected from an aminoacid having a positively charged side chain; X4 is selected from anamino acid having a positively charged side chain; and X5 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain. Some embodiments provide apeptide wherein X₁ is V or R; X2 is S, A, or H; X3 is K or R; X₄ is K orR; and X₅ is R, L, or K.

Some embodiments of this aspect provide for a peptide of Formula II:

X1-X2-X3-X4-X5-X6-X7-X8-X9  (SEQ ID NO:17);

wherein X1, X2, X3, X4, and X5 are as noted above, and each of X6, X7,X8, and X9 are independently selected from any amino acid, and areoptionally absent. In such embodiments, the peptide of Formula II caninclude 5 amino acid residues, 6 amino acid residues, 7 amino acidresidues, 8 amino acid residues, or 9 amino acid residues.

In another aspect the disclosure provides a peptide of Formula I:

X1-X2-X3-X4-X5  (SEQ ID NO:1)

or a salt thereof, wherein X1 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain; X2 is selected from an amino acid having a hydrophobic sidechain, an amino acid having a positively charged side chain, or an aminoacid having a polar uncharged side chain; X3 is selected from an aminoacid having a positively charged side chain; X4 is selected from anamino acid having a positively charged side chain; and X5 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain. Some embodiments provide apeptide wherein X₁ is V or R; X2 is S, A, or H; X3 is K or R; X₄ is K orR; and X₅ is R, L, or K.

A number of non-limiting embodiments of peptides according to Formula Iare disclosed in Table 1. In some embodiments the peptide can compriseVSRKR (SEQ ID NO:2), VSKRR (SEQ ID NO:3), VSRRR (SEQ ID NO:4), VARKL(SEQ ID NO:5), RHKKL (SEQ ID NO:6), RARRL (SEQ ID NO:7), RSKKL (SEQ IDNO:8), RHKRR (SEQ ID NO:9), VARRL (SEQ ID NO:10), VARRK (SEQ ID NO:11),or RSKRR (SEQ ID NO:12). In some embodiments, the peptide according toFormula I is VSRKR (SEQ ID NO:2), VSKRR (SEQ ID NO:3), VSRRR (SEQ IDNO:4), VARKL (SEQ ID NO:5), RHKKL (SEQ ID NO:6), RARRL (SEQ ID NO:7),RSKKL (SEQ ID NO:8), RHKRR (SEQ ID NO:9), VARRL (SEQ ID NO:10), VARRK(SEQ ID NO:11), or RSKRR (SEQ ID NO: 12).

In some embodiments of all the aspects described herein, the peptidesconsist essentially of the amino acid sequences and formulae disclosedherein. In some embodiments of the all aspects described herein, thepeptides consist of the amino acid sequences and formulae disclosedherein.

TABLE 1 Peptide Sequence SEQ ID NO: 1 X₁ X₂ X₃ X₄ X₅ SEQ ID NO: 2 V S RK R SEQ ID NO: 3 V S K R R SEQ ID NO: 4 V S R R R SEQ ID NO: 5 V A R K LSEQ ID NO: 6 R H K K L SEQ ID NO: 7 R A R R L SEQ ID NO: 8 R S K K L SEQID NO: 9 R H K R R SEQ ID NO: 10 V A R R L SEQ ID NO: 11 V A R R K SEQID NO: 12 R S K R R

In some embodiments, the peptides can exhibit at least one ApoE mimeticactivity. In some embodiments, for example, the disclosed peptides canbind one or more physiological ApoE receptors such as, for example,cell-surface receptors expressed by glial cells, as well as receptorsthat function to suppress the neuronal cell death and calcium influx(excitotoxicity) associated with N-methyl-D-aspartate (NMDA) exposure;protect against LPS-induced production of TNF-α and IL-6 (e.g., in an invivo sepsis model); prevent, treat, or slow inflammatory disorders suchas atherosclerosis, arthritis, or inflammatory bowel disease; suppressglial or microglial activation; suppress macrophage activation; suppresslymphocyte activation; suppress inflammation; suppress CNS inflammation;treat neuropathy; and/or ameliorate neuronal injury in neurodegenerativedisease (e.g., mild cognitive impairment, dementia, Parkinson's disease,or Alzheimer's disease) and/or acute CNS trauma (e.g., traumatic braininjury).

In some embodiments, the peptides will bind to a particular receptorwith similar affinity as ApoE. In some embodiments, the peptides willbind to a particular receptor with similar affinity as the previouslydisclosed longer 20-amino acid ApoE mimetic peptide which bindsmacrophages with a dissociation constant (K_(d)) of approximately 50 nM(Misra et al., (2001) J. Leukocyte Biol. 70:677-683). For example, thepeptide may bind to a receptor with a K_(d) equal or less than about 100μM, about 90 μM, about 80 μM, about 70 μM, about 60 μM, about 50 μM,about 40 μM, about 30 μM, about 20 μM, about 10 μM, about 5 μM, about 1μM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM,about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 5nM, about 1 nM, about 100 μM, about 90 μM, about 80 μM, about 70 μM,about 60 μM, about 50 μM, about 40 μM, about 30 μM, about 20 μM, about10 μM, about 5 μM, or about 1 μM. The peptide may bind to a receptorwith a K_(d) greater or equal than about 1 μM, about 5 μM, about 10 μM,about 20 μM, about 30 μM, about 40 μM, about 50 μM, about 60 μM, about70 μM, about 80 μM, about 90 μM, about 100 μM, about 1 nM, about 5 nM,about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 1 μM,about 5 μM, about 10 μM, about 20 μM, about 30 μM, about 40 μM, about 50μM, about 60 μM, about 70 μM, about 80 μM, about 90 μM, or about 100 μM.The peptide may bind to a receptor with a K_(d) in the range of about 1μM to about 10 μM, about 5 μM to about 15 μM, about 10 μM to about 20μM, about 20 μM to about 30 μM, about 30 μM to about 40 μM, about 40 μMto about 50 μM, about 50 μM to about 60 μM, about 60 μM to about 70 μM,about 70 μM to about 80 μM, about 80 μM to about 90 μM, about 90 μM toabout 100 μM, about 100 μM to about 1 nM, about 1 nM to about 10 nM,about 5 nM to about 15 nM, about 10 nM to about 20 nM, about 20 nM toabout 30 nM, about 30 nM to about 40 nM, about 40 nM to about 50 nM,about 50 nM to about 60 nM, about 60 nM to about 70 nM, about 70 nM toabout 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM,about 100 nM to about 1 μM, about 1 μM to about 10 μM, about 5 μM toabout 15 μM, about 10 μM to about 20 μM, about 20 μM to about 30 μM,about 30 μM to about 40 μM, about 40 μM to about 50 μM, about 50 μM toabout 60 μM, about 60 μM to about 70 μM, about 70 μM to about 80 μM,about 80 μM to about 90 μM, about 90 μM to about 100 μM, about 100 μM toabout 1 μM. For example, the peptide may bind to a macrophage with aK_(d) less than or equal to about 100 μM, about 90 μM, about 80 μM,about 70 μM, about 60 μM, about 50 μM, about 40 μM, about 30 μM, about20 μM, about 10 μM, about 5 μM, about 1 μM, about 100 nM, about 90 nM,about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about30 nM, about 20 nM, about 10 nM, about 5 nM, about 1 nM, about 100 μM,about 90 μM, about 80 μM, about 70 μM, about 60 μM, about 50 μM, about40 μM, about 30 μM, about 20 μM, about 10 μM, about 5 μM, or about 1 μM.

The extent of binding to an ApoE receptor can be assessed using anytechnique known in the art such as, for example, typical binding assays(e.g., competitive binding assays), ELISA, functional assays (e.g., asillustrated in the Examples), and the like. In some embodiments, thesize of the peptides can confer improved pharmacokinetics, facilitatecrossing the blood-brain barrier, allow intranasal administration,reduce production costs, increase potency (e.g., on a per-gram basis),and/or reduce peptide immunogenicity.

The peptides can be produced using any means for making polypeptidesknown in the art, including, e.g., synthetic and recombinant methods.For example, in some embodiments the peptides can be synthesized usingsynthetic chemistry techniques such as solid-phase synthesis,Merrifield-type solid-phase synthesis, t-Boc solid-phase synthesis, Fmocsolid-phase synthesis, BOP solid-phase synthesis, and solution-phasesynthesis. See, for example, Stewart and Young, Solid Phase PeptideSynthesis, 2^(nd) ed., (1984) Pierce Chem. Co., Rockford Ill.; ThePeptides: Analysis, Synthesis, Biology, Gross and Meienhofer, Eds.,vols. 1-2 (1980) Academic Press, New York; Bodansky, Principles ofPeptide Synthesis, (1984) Springer-Verlag, Berlin. In other embodiments,the peptides can be produced, for example, by expressing the peptidefrom a nucleic acid encoding the peptide in a cell or in a cell-freesystem according to recombinant techniques familiar to those of skill inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Current Protocols in Molecular Biology, (2002)John Wiley & Sons, Somerset, N.J.; each of which are hereby incorporatedby reference in their entireties. The peptides can incorporate any ofthe various modifications and protective groups described herein orotherwise known to those of skill in the art, such as, for example,those described in McOmie, Protective Groups in Organic Chemistry,(1973) Plenum Press, New York.

In some embodiments, the peptides can be designed to mimic physical,chemical, and/or structural features of the α-helical receptor-bindingdomain that lies between residues 130-150 of the native ApoE polypeptide(SEQ ID NO: 14). For example, in some embodiments the peptides can bedesigned to mimic physical, chemical, and/or structural features of thepolar face that extends along an outer surface of the three-dimensionalstructure of the native ApoE receptor-binding helix using a “linearwalk” peptide-design approach, in which the amino acid at eachsuccessive position in the peptide is selected by attempting to emulateone or more properties (e.g., relative size/steric hindrance, polar,non-polar, charged, uncharged, hydropathy index (e.g., hydrophobicity,hydrophilicity), acidic, basic, ability to form bonds (e.g., covalentbonds, hydrogen bonds, van der Waals interactions), etc.) of eachsuccessive residue exposed on the polar face of the native ApoEreceptor-binding helix by each descending periodic turn of the helix. Insome embodiments, the peptide may be designed based on the physical,chemical, and/or structural features of the ApoE-binding domain of oneor more ApoE receptors. For example, for an ApoE receptor having abinding pocket or surface that interacts with ApoE in an intermolecularreceptor-ligand binding interaction, the peptide may be designed tomaximize predicted binding affinity between the peptide and the ApoEbinding pocket/surface of the receptor by selecting peptide amino acidresidues expected to exhibit binding interactions along the receptor'sApoE binding pocket/surface.

Particular embodiments of peptide active agents as described above(shown in the form of their chemical structures), and for use in themethods and compositions described herein include, but are not limitedto, the following, along with pharmaceutically acceptable salts thereof:

In an aspect, the disclosure provides a method of treating aneurological condition in a subject in need thereof, the methodcomprising administering to the subject an effective amount of a peptideof Formula I or Formula II, or a composition or formulation comprisingan effective amount of a peptide of Formula I or Formula II, or acombination thereof.

In another aspect, the disclosure provides a method of reducinginflammation in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a peptide of FormulaI or Formula II, or a composition or formulation comprising an effectiveamount of a peptide of Formula I or Formula II, or a combinationthereof.

In embodiments relating to the above described aspects, the peptidesand/or compositions can be used to treat, ameliorate, or prevent certainsigns, symptoms, and/or deleterious neurological effects of acute and/orchronic CNS injury. As used herein, acute CNS injury includes but is notlimited to stroke (caused by thrombosis, embolism or vasoconstriction),closed head injury, traumatic brain injury, global cerebral ischemia(e.g., ischemia due to systemic hypotension of any cause, includingcardiac infarction, cardiac arrhythmia, hemorrhagic shock, and postcoronary artery bypass graft brain injury), ischemic stroke, globalanoxia, focal ischemia, subarachnoid hemorrhage, and intracranialhemorrhage. Ischemic damage to the central nervous system may resultfrom either global or focal ischemic conditions. Global ischemia occurswhere blood flow to the entire brain ceases for a period of time, suchas during cardiac arrest. Focal ischemia occurs when a portion of thebrain is deprived of normal blood flow, such as during thromboembolyticocclusion of a cerebral vessel, traumatic head injury, edema and braintumors. Much of the CNS damage due to cerebral ischemia occurs duringthe hours or even days following the ischemic condition, and issecondary to the release of cytotoxic products by damaged tissue.Chronic CNS injury includes but is not limited to Alzheimer's disease(AD), Parkinson's disease, epilepsy, and HIV-associated encephalopathy.The finding that ApoE peptides may suppress glial activation provides arole for disclosed methods, peptides, and compositions treating anyneurological disease involving microglial activation. For example,microglia express markers of activation in AD, suggesting that crucialinflammatory events in AD involve microglia. Such activated microgliacluster near amyloid plaques. Microglia are also activated in epilepsy.

In some embodiments, the peptides and/or compositions can be used toprevent, treat, or ameliorate the clinical neurological signs andsymptoms associated with inflammatory conditions affecting the nervoussystem (e.g., the CNS). Non-limiting examples include multiplesclerosis, vasculitis, acute disseminated encephalomyelitis, andGuillain-Barre syndrome. In this regard, the disclosed ApoE mimeticpeptides can be used alone or in combination with other knownanti-inflammatory drugs or cytokines to formulate pharmaceuticalcompositions for the treatment of CNS inflammatory conditions.

In some embodiments, the peptides and/or compositions can be used toprevent, treat, or ameliorate conditions associated with NMDAexcitotoxicity. NMDA excitotoxicity has been associated withneurolathyrism, amyotrophic lateral sclerosis (ALS), schizophrenia, HIVdementia and encephalopy, Huntington's chorea, Parkinson's disease,bipolar disorder, multiple sclerosis in humans and experimental allergicencephalomyelitis (EAE) in animals, pain, depression, stroke, epilepsy,inherited d-2-hydroxyglutaric aciduria, AD, and traumatic brain injury.In some embodiments, the peptides and/or compositions can block NMDAreceptor mediated excitotoxicity and provide neuroprotection. NMDAantagonists are also used in clinical anesthesia, and have been shown toinhibit chronic pain, drug tolerance, and alcohol dependency. Thus, insome embodiments, the disclosed methods, peptides, and compositions maybe used as ingredients in anesthesia formulations and in combinedtherapeutic compositions containing other known compounds useful fortreating the described conditions.

In some embodiments, the peptides and/or compositions can be used toprotect against LPS-induced production of cytokines in sepsis. IntactApoE has been shown to protect mice from bacterial LPS-inducedlethality. Other possible sepsis co-therapies involve administeringanti-inflammatory cytokines, including IL-10, transforming growthfactor-beta, granulocyte colony-stimulating factor, IFN-phi, macrophagemigration inhibitory factor and high mobility group 1 protein, andmonoclonal antibodies, including anti-endotoxin antibodies, anti-tumornecrosis factor antibodies, and anti-CD14 antibodies. Thus, embodimentsprovide for the use of peptides alone or in combination with other knownanti-inflammatory cytokines and antibodies in compositions and methodsfor treating sepsis.

The effect of the disclosed methods, peptides, and compositions may beassessed at the cellular or tissue level (e.g., histologically ormorphometrically) or by assessing a subject's neurological status. Thesuppression or reduction of glial activation can be assessed by variousmethods as would be apparent to those in the art; one such method is tomeasure the production or presence of compounds that are known to beproduced by activated glia, and compare such measurements to levels ofthe same compounds in control situations. Alternatively, the effects ofthe present methods and compounds in suppressing, reducing or preventingmicroglial activation may be assessed by comparing the signs and/orsymptoms of CNS disease in treated and control subjects, where suchsigns and/or symptoms are associated with or secondary to activation ofmicroglia.

Typically, the terms “treating” and “treatment” when used with referenceto a disease or a subject in need of treatment includes, but is notlimited to, halting or slowing of disease progression, remission ofdisease, prophylaxis or lessening of symptoms and/or clinicalindications, reduction in disease and/or symptom severity, or reductionin disease length as compared to an untreated subject, and/or in theabsence of treatment. In embodiments, the methods of treatment can abateor ameliorate one or more clinical indications of the particular diseasebeing treated. Certain embodiments relating to methods of treating adisease or condition associated with an ApoE activity compriseadministration of therapeutically effective amounts of a peptide ofFormula I, or of Formula II, or one or more peptides selected from SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, as well as pharmaceutical compositions thereof. In embodiments,the method of treating can relate to any method that prevents furtherprogression of the disease and/or symptoms, slows or reduces the furtherprogression of the disease and/or symptoms, or reverses the diseaseand/or clinical symptoms associated with ApoE activity.

Subjects to be treated by the methods described herein encompassmammalian subjects, including both human subjects and non-human (animal)subjects such as dogs, cats, rabbits, goats, horses, pigs, cattle, etc.(including both male and female subjects and subjects of all agesincluding infant, juvenile, adolescent and adult subjects). Subjects maybe treated for any purpose, such as for reducing inflammation,suppressing microglial activation, ameliorating chronic disease, etc.The term “concurrently administered” as used herein means that twocompounds are administered sufficiently close in time to achieve acombined immunological effect. Concurrent administration may thus becarried out by sequential administration or simultaneous administration(e.g., simultaneous administration in a common, or the same, carrier).

In some embodiments, the disclosed peptides and compositions may beadministered by any suitable route of administration, including, but notlimited to, injection (subcutaneous, intraperitoneal, intravenous,intrathecal, intramuscular, intracerebroventricular, and spinalinjection), intranasal, oral, transdermal, parenteral, inhalation,nasopharyngeal or transmucosal absorption. Administration encompassesthe providing at least one peptide as described herein (e.g., of FormulaI, Formula II, and/or SEQ ID NOs:1-12) formulated as a pharmaceuticalcomposition. Administration of an active agent (e.g., compound, peptide,etc.) directly to the brain is known in the art. Intrathecal injectiondelivers agents directly to the brain ventricles and the spinal fluid.Surgically-implantable infusion pumps are available to provide sustainedadministration of agents directly into the spinal fluid. Spinalinjection involves lumbar puncture with injection of a pharmaceuticalcompound into the cerebrospinal fluid. Administration also includestargeted delivery wherein peptide according to the disclosure is activeonly in a targeted region of the body (for example, in brain tissue), aswell as sustained release formulations in which the peptide is releasedover a period of time in a controlled manner. Sustained releaseformulations and methods for targeted delivery are known in the art andinclude, for example, use of liposomes, drug loaded biodegradablemicrospheres, drug-polymer conjugates, drug-specific binding agentconjugates and the like. Pharmaceutically acceptable carriers are wellknown to those of skill in the art and include chitosan nanoparticles orother related enteric polymer formulations. Determination of particularpharmaceutical formulations and therapeutically effective amounts anddosing regimen for a given treatment is within the ability of one ofskill in the art taking into consideration, for example, patient age,weight, sex, ethnicity, organ (e.g., liver and kidney) function, theextent of desired treatment, the stage and severity of the disease andassociated symptoms, and the tolerance of the patient for the treatment.

Some embodiments of the methods described herein provide for intranasaldelivery of one or more peptides described herein, or a compositioncomprising a peptide, for a subject having a chronic disease such as,for example multiple sclerosis, Alzheimer's disease, epilepsy,Parkinson's disease, arthritis, inflammatory bowel disease, leukemia, oratherosclerosis. Formulations and methods appropriate for intranasal andinhaled administration are known in the art.

In embodiments relating to therapeutic applications, the administrationcan be performed on a subject already suffering from the disorder ofinterest. Those in the incubation phase or the acute phase of thedisease can be treated by the methods described herein, either alone orin conjunction with other treatments, as suitably based on theparticular disease/condition, patient, and combination. One of skill inthe art will be able to determine when a combination treatment is or isnot suitable.

In therapeutic methods and uses, the peptides and composition describedherein can be administered to a subject in an amount sufficient totreat, or at least partially arrest, symptoms and/or complications. Anamount adequate to accomplish this is often referred to as“therapeutically effective dose.” Amounts effective for this use willdepend in part on the peptide, composition, the manner ofadministration, the stage and severity of the condition being treated,the age, weight, and general health of the patient, and the judgment ofthe prescribing physician.

In embodiments, effective amounts of the compositions and peptidesdisclosed herein can include less than about 100 mg/kg, less than about50 mg/kg, less than about 25 mg/kg, less than about 10 mg/kg, less thanabout 1 mg/kg, less than about 0.1 mg/kg, less than about 0.05 mg/kg,less than about 0.01 mg/kg, less than about 0.005 mg/kg, and less thanabout 0.001 mg/kg peptide. In some embodiments, effective amounts of thecompositions and peptides disclosed herein can include at least about0.0001 mg/kg, at least about 0.001 mg/kg, at least about 0.005 mg/kg, atleast about 0.01 mg/kg, at least about 0.05 mg/kg, at least about 0.1mg/kg, at least about 0.5 mg/kg, at least about 1 mg/kg, at least about5 mg/kg, and at least about 10 mg/kg peptide. This includes, forexample, peptide amounts ranging from about 0.0001 mg/kg to about 100mg/kg, from about 0.001 mg/kg to about 10 mg/kg, from about 0.005 mg/kgto about 0.5 mg/kg, and from about 0.01 mg/kg and about 0.05 mg/kg. Insome embodiments, the methods, peptides, and compositions describedherein can be employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, it is possible and may be felt desirable by the treatingphysician to administer substantial excesses of these compositions.Additionally, one of ordinary skill in the art would also know how toadjust or modify variables such as dosage, dosage schedules, and routesof administration, as appropriate, for a given subject.

The disclosed peptides and compositions may be administered acutely(i.e., during the onset or shortly after events leading to the conditionrequiring treatment), prophylactically (e.g., before scheduled surgery,or before the appearance of neurologic signs or symptoms), or during thecourse of a degenerative disease to reduce or ameliorate the progressionof symptoms that would otherwise occur. The timing and interval ofadministration is varied according to the subject's symptoms, and may beadministered at intervals spanning minutes, hours, or days, over a timecourse of hours, days, weeks or longer, as would be determined by oneskilled in the art.

Some embodiments relating to pharmaceutical compositions for therapeuticor prophylactic treatment provide for formulations specific for any ofmucosal (oral, nasal, inhalation, rectal, vaginal, tracheal, etc.),parenteral, topical, or local administration. For purposes herein,mucosal administration is different from topical administration, asmucosal administration refers to application of the vaccine to a mucosalsurface such as a surface of the respiratory tract, gastrointestinaltract, reproductive tract, etc. In some embodiments, the pharmaceuticalcompositions are suitably administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly.Topical administration (i.e., non-mucosal) can be to a non-mucosalsurface of a subject, such as the eye, ear, nails, hair, or skin, in anyappropriate form such as aqueous or non-aqueous liquid (e.g., droplet),emulsion, paste, ointment, cream etc. Thus, the disclosure providescompositions for topical (mucosal or non-mucosal) or parenteraladministration which comprise one or more small ApoE mimetic peptides,dissolved or suspended in an acceptable carrier, such as an aqueouscarrier. In embodiments, the pharmaceutical composition is administerednasally. Any variety of aqueous carriers may be used, e.g., water,buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.These compositions can be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingsolutions may be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc. Alternatively, the pharmaceuticalcompositions described herein can also be in dry powder formulations. Inembodiments relating to dry powder vaccine formulations, typically theliquid vaccine is rapidly frozen and dried in a vacuum (e.g.,freeze-dried) in the presence of at least one bulking agent (such astrehalose or other sugars) to provide a vaccine formulation that hassuperior temperature stability. Such dry powder vaccine formulations maybe administered to the host as a dry powder, thereby eliminating theneed for liquid reconstitution.

In aspects described herein that relate to compositions, includingpharmaceutical compositions and formulations, some embodiments provide acomposition that comprises at least one peptide according to SEQ ID NO:1(e.g., SEQ ID NOs:1-12), in combination with an acceptable carrier,vehicle, diluent, or adjuvant. In further embodiments the compositioncomprises a peptide selected from any of SEQ ID NOs:2-12, or anycombinations of two or more peptides thereof, in combination with acarrier, vehicle, diluent, or adjuvant.

In some embodiments, the disclosure provides a composition that consistsessentially of a peptide of SEQ ID NO:1 or of SEQ ID NO:17 and acarrier, vehicle, diluent, or adjuvant. In further embodiments thecomposition consists essentially of a peptide selected from any of SEQID NOs:2-12, or any combinations of two or more peptides thereof, and acarrier, vehicle, diluent, or adjuvant.

In an aspect, the disclosure provides a medicament for treating aneurological condition in a subject in need thereof, wherein themedicament comprises an effective amount of a peptide of Formula Iand/or Formula II.

In an aspect, the disclosure provides a medicament for treatinginflammation in a subject in need thereof, wherein the medicamentcomprises an effective amount of a peptide of Formula I and/or FormulaII.

While the following examples provide further detailed description ofcertain aspects and embodiments of the disclosure, they should beconsidered merely illustrative and not in any way limiting to the scopeof the claims.

EXAMPLES Example 1: Materials and Methods for Cell Culture-BasedAssessment of Glial Activation

In vitro methods have been developed using cultured cells for evaluatingthe ability of a compound, such as a peptide, to suppress glialactivation. Laskowitz et al., (2001) Exp. Neurol., 167:74-85. Glial cellcultures were grown and maintained under standard conditions. Culturedglial cells may include primary murine mixed glial cell cultures, murineBV-2 microglial cells, and human C3 microglial cells. Adherent glialcells were washed with OptiMEM® medium (available from Invitrogen Corp.)to remove serum and covered with fresh OptiMEM® medium containingpeptide.

Peptides of 5 amino acids in length were applied to one or more samplesof cells in parallel at concentrations ranging from about 0 to about 50μM, such as 0.3, 3, and 30 μM. The peptides AL-10-1 (SEQ ID NO:18) andVL-17-9 (SEQ ID NO:19), which are longer peptides (12 amino acids each)were used as negative and positive controls, respectively. A series ofpeptides were screened in primary rat neuronal cortical culture exposedto NMDA using the method described in Aono et al., Neuroscience (2003)116:437 and Aono et al., Neurobiology of Disease (2002) 11:214, both ofwhich are incorporated by reference in their entirety (see Table 2).Neuroprotection from NMDA mediated cell death in primary neuronalculture was expressed as percentage decrease in LDH, 24 hours afterexposure to NMDA compared to vehicle treated cultures (Table 2). LDHrelease is indicative of neuronal death after NMDA exposure. The peptideVR-55 (SEQ ID NO:4) reduced NMDA mediated excitotoxic cell death byapproximately 19% at a concentration of 1 μM. Effects were specific, andnot all peptides demonstrated neuroprotection. The positive controlpeptide VL-17-9 reduced LDH release by 31%. In comparison, the shorterVL-5-2 reduced LDH release by 31%, indicating that the shorter peptidesalso reduce NMDA mediated excitotoxic cell death.

TABLE 2 0.1 μM 0.3 μM 1 μM 3 μM VR-55 Ac-VSRRR-NH2 (SEQ ID NO: 4) −2.85%    1.50% 19.14% 15.91% VR-54 Ac-VSKKR-NH2 (SEQ ID NO: 13)−15.91% VR-53 Ac-VSKRR-NH2 (SEQ ID NO: 3) 7.03% VR-52 Ac-VSRKR-NH2 (SEQID NO: 2) 12.63% RL-5-3 Ac-RSKKL-NH2 (SEQ ID NO: 8) −20.80% RL-5-2Ac-RARRL-NH2 (SEQ ID NO: 7) −5.23% RL-5-1 Ac-RHKKL-NH2 (SEQ ID NO: 6)−2.85% RR-5-2 Ac-RSKRR-NH2 (SEQ ID NO: 12) 0.08% RR-5-1 Ac-RHKRR-NH2(SEQ ID NO: 9) 7.50% VL-5-3 Ac-VARRL-NH2 (SEQ ID NO: 10) −19.04% −16.70%11.50% VL-5-2 Ac-VARKL-NH2 (SEQ ID NO: 5) 34.26% VL-5-1 Ac-VARKL-NH2(SEQ ID NO: 5)   18.57%    0.29% 24.48% AL-10-1 Ac-ASHLRKLRKRLL-NH2 (SEQID NO: 18) −6.21% VL-17-9 Ac-LRVRLASLLRKL-NH2 (SEQ ID NO: 19)   16.19%30.95%

Dose response of the neuroprotection from NMDA excitotoxicity in primaryneuronal culture was determined for ApoE mimetic peptide VR-55 (SEQ IDNO: 4). FIG. 6 shows that the percent suppression of LDH release as afunction of exposure to VR-55 (SEQ ID NO: 4) is dose dependent. Table 3shows the cumulative data of all of the peptides in the related familythat were screened in the same bioassay for their suppressioncapabilities, all at a concentration of 1 μM. “0” indicates less than 5%suppression; “+” indicates 5-10% suppression; “++” indicates 11-20%suppression; and “+++” indicates greater than 20% suppression. Inaddition to VL-17-9, several other candidates had greater than 20%suppression, including VR-55, VL-5-1, VL-5-2 and VR-52.

TABLE 3 List of ApoE mimetic peptides Peptide 1 μM VR-52 (SEQ ID NO: 2)+++ VR-53 (SEQ ID NO: 3) + VR-54 (SEQ ID NO: 13) ++ VR-55 (SEQ ID NO: 4)+++ RL-5-1 (SEQ ID NO: 6) ++ RL-5-2 (SEQ ID NO: 7) 0 RL-5-3 (SEQ ID NO:8) 0 RR-5-1 (SEQ ID NO: 9) + RR-5-2 (SEQ ID NO: 12) 0 VL-5-1 (SEQ ID NO:5) +++ VL-5-2 (SEQ ID NO: 5) +++ VL-5-3 (SEQ ID NO: 10) ++ AL-10-1 (SEQID NO: 18) 0 VL-17-9 (SEQ ID NO: 19) +++

Larger peptides or proteins may need to be tested at higherconcentrations to observe measurable suppression of glial activation. Ifusing primary murine cells and BV-2 cells, cells are stimulated with 100ng/ml E. coli LPS (available from Sigma-Aldrich Co.), and supernatant iscollected six hours after LPS stimulation and assayed for nitrite (usinga colorimetric Greiss reagent system, available from Promega Corp.)and/or TNF-α (using a solid-phase ELISA kit, available from InvitrogenCorp.). If using human C3 cells, cells are stimulated with 200 μg/mlpolyinosinic acid 5′ (available from Sigma-Aldrich Co.), and supernatantis collected 5 days after stimulation and assayed for TNF-α using asolid-phase ELISA kit (available from Invitrogen Corp.).

Example 2: Suppression of Microglial Activation by ApoE Mimetic Peptide

Murine BV-2 cultures were prepared and used to evaluate suppression ofmicroglial activation as described in Example 1. Replicate samples ofBV-2 cells were incubated without peptide, with an ApoE mimetic peptide(VSKRR; SEQ ID NO:3) at either 0.3 μM, 3 μM, or 30 μM, or with anegative control peptide (VSKKR; SEQ ID NO:13) at either 0.3 μM, 3 μM,or 30 μM. Each sample was stimulated with LPS and assessed for TNF-αproduction as described in Example 1. As depicted in FIG. 1, treatmentwith ApoE mimetic peptide (SEQ ID NO:3) at each dosage tested resultedin decreased TNF-α production relative to untreated cells or cellstreated with negative control peptide (SEQ ID NO:13). The data indicatethat peptides disclosed herein were useful in reducing the release ofpro-inflammatory mediators.

The protective activity against brain injury-downregulating CNSinflammatory response was determined for ApoE mimetic peptides, VR-55(SEQ ID NO: 4), VL-5-1 (SEQ ID NO: 5) and VL-5-3 (SEQ ID NO: 10). FIGS.11A and 11B show that VR-55, VL-5-1, and VL-5-3 suppress the release ofinflammatory cytokine TNF-α in mixed glial primary culture afterexposure to LPS.

Example 3: Materials and Methods for Testing Neurological Deficits InVivo

An experimental murine model for traumatic brain injury was developed.Laskowitz, et al. (2010) J. Neurotrauma, 27:1983-95. Male mice (age12-14 weeks) were anesthetized with 4.3% isoflurane in oxygen in ananesthesia induction box for 90 seconds. The trachea was intubated andthe lungs mechanically ventilated with 1.4% isoflurane in a 50/50mixture of oxygen and nitrogen. Body temperature was maintained at 37°C. using surface heating/cooling. The top of the skull was exposed by amidline incision to identify anatomical landmarks, and a concave 3-mmmetallic disc was secured to the skull surface with an adhesive,directly midline and just caudal to the bregma. The disc diffused theenergy of impact and reduced the incidence of depressed skull fractureto less than 10%. After general anesthesia, mice were positioned in astereotactic device and the skull was exposed. A pneumatic impactor(diameter: 2.0 mm; available from Air-Power, Inc.) discharged at 6.8±0.2m/s with a head displacement of 3 mm was used to deliver a singlemidline impact to the disc surface.

An experimental murine model for intracerebral hemorrhage was alsoprovided. James, et al. (2009) Stroke, 40:632-39. Male mice (age 16-20weeks) were anesthetized with 4.6% isoflurane in oxygen in an anesthesiainduction box for 90 seconds. The trachea was intubated and the lungsmechanically ventilated with 1.6% isoflurane in a 70/30 mixture ofnitrogen/oxygen. Body temperature was maintained at 37° C. using anunderbody warming system. The animal's head was secured in astereotactic frame, local anesthetic was injected, and the scalpincised. After exposure of the skull, a burr hole was created 2 mm leftlateral to bregma, and a 0.5 μL syringe needle (available from HamiltonCo.) was advanced to a depth of 3 mm from cortex. Type IV-S clostridialcollagenase (available from Sigma-Aldrich Co.) was injected over aperiod of 5 minutes (0.1 U in 0.4 μL normal saline). The incision wasthen closed, and animals were allowed to recover spontaneous ventilationwith subsequent extubation.

An experimental procedure for measuring neurological deficits wasdeveloped. An automated rotorod (available from Ugo Basile NorthAmerica, Inc.) was used to assess vestibulomotor function. On the daybefore experimental induction of a neurological condition or injury(such as, for example, traumatic brain injury or intracerebralhemorrhage as described above), mice under went two consecutiveconditioning trials at a set rotational speed (16 revolutions perminute) for 60 seconds followed by three additional trials with anaccelerating rotational speed. The average time to fall from therotating cylinder in the latter three trials was recorded as baselinelatency. After injury, mice under went consecutive daily testing withthree trials of accelerating rotational speed (intertribal interval of15 minutes). Average latency to fall from the rod was recorded, and miceunable to grip the rod were scored with a latency of 0 seconds.

Another experimental procedure for measuring neurological deficits wasdeveloped using a Morris water maze. The procedure used a black aluminumpool containing a movable platform (7.5 cm diameter) and filled with25-27° C. water opacified with powdered milk. Each training or testingsession consisted of four trials per day with an interval of 20-30minutes between trials. One day before testing, mice were trained usinga visible platform (platform flagged and located in a different quadranteach trial to minimize quadrant habituation, no extra-maze visual cues)to habituate the mice to handling and swimming and to teach the mice thegoal of the test-escaping the water by climbing on the platform. Afterthe training day, mice were tested with a hidden platform submerged 1 cmbelow the water surface (four consecutive days, platform submerged inwestern quadrant for all trials with several extra-maze visual cues).For each test, mice were placed in the pool facing the perimeter andallowed to search for the platform for a maximum of 90 seconds. Micewere started in one of four different quadrants for each trial, with thestarting quadrant order randomly defined each day. Latency to findingthe platform and swimming speed were recorded using a computerized videotracking system (Ethovision 2.2.14; available from Noldus InformationTechnology, Leesburg Va.).

Example 4: Effect of ApoE Mimetic Peptide on Neurological Outcomes afterTraumatic Brain Injury

Groups of mice received either an ApoE mimetic peptide (SEQ ID NO:3;0.05 mg/kg or 0.2 mg/kg) or carrier (saline) by intravenous tail veininjection at 2 hours and again at 6 hours after traumatic brain injuryinduced as described in Example 3. Each mouse was tested forvestibulomotor function by rotorod latency measured before and afterinjury, as described in Example 3. Animals treated with 0.05 mg/kg or0.2 mg/kg ApoE mimetic peptide (SEQ ID NO:3) showed significantlyimproved vestibulomotor performance compared to carrier treatment asreflected by increased rotorod latency. See FIG. 2. The effect wasdurable through the five-day testing period. The data indicate thatpeptides disclosed herein are useful in treating traumatic brain injury.

Example 5: Effect of ApoE Mimetic Peptide on Neurological Outcomes afterTraumatic Brain Injury

Groups of mice received either an ApoE mimetic peptide (SEQ ID NO:4;0.05 mg/kg) or carrier (saline) by intravenous tail vein injection at 2hours and again at 6 hours after traumatic brain injury induced asdescribed in Example 3. Each mouse was tested for vestibulomotorfunction by rotorod latency measured before and after injury, asdescribed in Example 3. Animals treated with 0.05 mg/kg ApoE mimeticpeptide (SEQ ID NO:4) had improved vestibulomotor performance asreflected by increased rotorod latency. See FIG. 3. The effect wasdurable through the five-day testing period. Other groups of micereceived either an ApoE mimetic peptide (SEQ ID NO:4; 0.05 mg/kg or 0.2mg/kg) or carrier (saline) by intravenous tail vein injection at 2 hoursand again at 6 hours after traumatic brain injury induced as describedin Example 3. Each mouse was tested for neurocognitive performance ondays 28, 29, 30, and 31 post-injury using a Morris water maze asdescribed in Example 3. Animals treated with ApoE mimetic peptide (SEQID NO:4) at either 0.05 mg/kg or 0.2 mg/kg exhibited improvedneurocognitive outcomes as reflected by water maze latency. See FIG. 4.The data indicate that peptides disclosed herein are useful in treatingtraumatic brain injury.

Example 6: Effect of ApoE Mimetic Peptide on Neurological Outcomes afterIntracerebral Hemorrhage

Groups of mice received either an ApoE mimetic peptide (SEQ ID NO:4;0.05 mg/kg) or carrier (saline) by intravenous tail vein injection a 2hours and again at 6 hours after intracerebral hemorrhage induced asdescribed in Example 3. Each mouse was tested for vestibulomotorfunction by rotorod latency measured before and after inducedintracerebral hemorrhage as described in Example 3. Animals treated with0.05 mg/kg ApoE mimetic peptide (SEQ ID NO:4) had improvedvestibulomotor performance as reflected by increased rotorod latency.See FIG. 5. The effect was durable through the five-day testing period.The data indicate that peptides disclosed herein are useful in treatingintracerebral hemorrhage.

Example 7: Effect of ApoE Mimetic Peptide on Neurological Outcomes afterBlast Injury Data

A blast injury mouse study was performed using a shock tube blast model.A set of three shock tubes (FIG. 7A) was built to provide a range ofblast conditions with realistic peak overpressure, scaled duration, andimpulse. For peptide testing, the 1240 mm length, 78 mm diameter shocktube was used. The driver section was constant for all tests, andconsisted of a 25 mm thick spacer flange bolted together with acorresponding blind flange and slip-on flange attached to the drivenpipe. This driver section profile may be varied to change theoverpressure characteristics of the tube. Full-faced gaskets(Graphite/Buna-N material) were installed between each flange to preventleakage. The diaphragm was composed of a number of sheets ofpolyethylene terephthalate (PET) film installed between the driverspacer flange and the flange attached to the driven section. The driversection was filled with high-pressure helium through a fitting on theback of the blind flange until the diaphragm ruptured, sending the shockwave down the driven tube section.

The shock tube was mounted vertically on an extruded aluminum frameusing three vibration-damping U-bolts. Three flush-mountedpiezoresistive pressure transducers (PT) (Endevco 8530B; Endevco Corp.,San Juan Capistrano, Calif.) were spaced 1200 around the diameter,offset 6 mm from the open end of the shock tube. Since the wallthickness of the tube was less than the length of the PT, a 6 mm thickcollar (19 mm long) fit over the end of the tube and was welded in placeto provide additional mounting support for the PTs. An additional PT wasinstalled in the driver section to measure the burst pressure when thediaphragm ruptured. An aluminum fixture was used to provide thoracicprotection for the mice (FIG. 7B). In previous testing, peakoverpressure and impulse were decreased by more than a factor of 10.Peak incident overpressure, positive-phase duration, and peak incidentimpulse were recorded in the three end-tube PTs for each test (data notshown). The level of driver burst pressure was controlled using a rangeof diaphragm thicknesses (0.58 to 0.84 mm), and the driver gas tankpressure was regulated to 7.0 MPa. Atmospheric conditions (temperature,barometric pressure, humidity) were recorded prior to each test. Allsensors were sampled at 1 MHz with a 500 kHz anti-aliasing filter. Datawas post-processed using an 8^(th)-order low-pass Butterworth filterwith a cutoff frequency of 40 kHz.

Wild-type mice (Jackson Labs) were exposed to blast in two groups of 15,one with peptide injection (SEQ ID NO:4), and one with vehicle-onlycontrols. The neurological deficits were measured in injured mice usingthe Morris water maze as described above. The efficacy of ApoE mimeticpeptide on neurocognitive performance was examined in mice with blastinjury as determined using the Morris water maze (FIG. 8). FIG. 8 showsthat ApoE mimetic peptide reduces neurocognitive deficit after blastinjury. Administration of the peptide resulted in a trend towardsenhanced cognitive performance as demonstrated by increased time in thequadrant with the previously learned hidden platform (i.e., a probetrial to evaluate retention capabilities as described in Laskowitz etal., J. Neurotrauma, 24:1093-1107 (2007))(data not shown). Vehicletreated animals spent 17.8±1.9 seconds in the correct quadrant comparedto the ApoE mimetic peptide (SEQ ID NO:4) treated animals which spent21.8±2.7 seconds in the correct quadrant, p=0.24. The trend was towardsimproved learning in the Morris water maze (p=0.07). The trend wastowards improved performance in the probe trial (p=0.25).

The mouse apnea data scales to other species and may be a good modelsystem. The blast injury model was unique from the blunt injury model inthe recovery of motor function and in the persistent and early cognitivedeficits. With the rotorod results, the blast mice appeared to regainmotor function quickly. There was no significant deficit between shamand injury condition post-blast. With the Morris water maze results, theblast mice showed significant cognitive deficits throughout the watermaze trials.

Example 8: Pharmacokinetics in Blood and CNS for Intravenous Delivery

The amount of the ApoE mimetic peptide VSRRR (SEQ ID NO:4) in bloodplasma and CNS was determined as follows:

VSRRR Quantitation in Mouse Plasma using LC/Selected Ion Monitoring(SIM)/MS

48 μL aliquots of mouse plasma were measured into the wells of a 2 mL96-well plate. 6 μL of a stable isotope labeled (SIL) form of the VSRRRpeptide (“VSRRR[10]”; SEQ ID NO:4) (5 picomoles/μL in 50 mM ammoniumbicarbonate, pH 8, buffer) was added. 6 μL of a synthetic form of theVSRRR peptide (“VSRRR”; SEQ ID NO:4) in 50 mM Ammonium bicarbonate wasadded for standards and quality controls (QCs) and an equivalent volumeof 50 mM ammonium bicarbonate was added to all wells containing mouse PKsamples. 1140 μL of 50 mM ammonium formate, pH 10, was added for a finalvolume of 1200 μL. Salts and proteins were removed using OASIS® HLBSolid Phase Extraction (SPE) protocol as follows:

1. 500 μL methanol (MeOH) through each well×1.

2. 500 μL 25% acetonitrile (ACN)/1% trifluoroacetic acid (TFA)×1 (aspre-elution).

3. 500 μL MeOH×1

4. 500 μL 50 mM ammonium formate×2

5. 1 mL of each sample mixture was pipetted directly onto an OASIS® HLBplate (hydrophilic-lipophilic-balanced reversed-phase sorbent; WatersCorp.) into a corresponding well and slowly vacuumed through

6. 500 μL 50 mM ammonium formate×1

7. 500 μL 10% ACN/50 mM ammonium formate×2

8. 500 μL 25% ACN/50 mM ammonium formate×1

9. Removed plate collecting washes/flowthrough, and put in collectionplate

10. Eluted with 100 μL 25% ACN/1% TFA×3, eluting with vacuum slowly. Thefinal eluate should be approximately 300 μL.

The SPE eluate was dried using a vacuum centrifuge. The sample wasreconstituted in 50 μL of buffer containing 1% acetonitrile (ACN), 0.1%trifluoroacetic acid (TFA), and 0.02% heptafluorobutyric acid (HFBA).Two microliters of reconstituted sample was analyzed by nanoscalecapillary LC coupled to a high resolution, accurate mass tandem massspectrometer. Specifically, the electrospray ionization source withNanoLockSpray™ (Waters Corp.) and nanoAcquity UPLC® system (WatersCorp.) were used with a nanoscale LC column (1.7 μm BEH130 C18 150 μmID×100 mm long; Waters Corp.), 10-min gradient of 3% to 19% ACN with0.1% formic acid (mobile phase A=0.1% formic acid/0.001% HFBA) with atotal LC run time of 16.5 minutes, flow rate=1.8 μL/min and 35° C.column temperature. A SYNAPT™ G1 HDMS™ high resolution mass spectrometer(Waters Corp.) was used. Full Scan MS data was obtained over the massregion of 50-4000 Da using an Enhanced Duty Cycle scan function at 360Da.

VSRRR and VSRRR[10] amounts were quantitated by measuring the area underthe curve (AUC) of the Selected Ion Chromatograms of the doubly chargedions at high resolution (m/z 357.7 and 362.7). The final VSRRRquantitation amount was determined using the ratio of the AUCs(VSRRR/VSRRR[10]). The ratios from 5 animals were averaged per timepoint and calibration standards were used to generate a standard curve.Duplicate aliquots of QC samples were analyzed by LC/MS in triplicate todetermine analytical reproducibility.

FIG. 9 shows the amount of peptide in the plasma sample over time aftera single dosing of 0.8 mg/kg of the peptide. The lower level ofquantitation (LLOQ) is indicated. Table 4 shows the results ofintravenous (IV).

TABLE 4 IV Ratio Light/Heavy Stdev % CV across 5 across 5 across 5 TimePoint (min) animals animals animals fmol/uL 1 0.079 0.030 37.8 32.2 30.134 0.045 33.4 51.8 5 0.077 0.054 69.4 31.7 10 0.058 0.024 41.1 24.915 0.044 0.012 26.9 19.6 30 0.016 0.004 25.7 9.9 60 0.012 0.008 64.7 8.5

Mouse Brain PK on Therapeutic Peptide, VSRRR—Sample PreparationProcedure

Mice brains were weighed in a 1.5 mL Eppendorf tube. The entire brainwas transferred to a 14 mL culture tube. 1 mL of 8 M urea in 50 mMammonium formate (pH 10) with 2.5 pmol/mL SIL peptide per 100 mg wettissue weight was added. For standard controls and QCs, 10 μL of peptidestandard per every 100 mg of brain tissue (1%) was added with 990 μL ofthe aforementioned buffer to bring to a 1 mL total volume for every 100mg of tissue. A tissue tearor was used on each sample for about 20 sec.1.5 mL of the sample was transferred to a 2-mL Eppendorf tube. Eachsample was probe sonicated for 3 bursts of 5 sec per burst. The samplewas then heated at 37° C. for 30 min. The sample was centrifuged for 30min at 15,000 rpm. Very little precipitate was visible at the bottom ofeach tube and avoided when pipetting 1 mL of the sample (out of 1.5 mLtotal volume) and placing directly onto an OASIS® plate. Salts andproteins were removed using the OASIS® HLB Solid Phase Extraction (SPE)protocol described above for the plasma sample. After the extraction,the samples were dried down in a Speed Vac and reconstitute in 25 μL of1% ACN/0.1% TFA/0.02% HFBA. 3 μL of the sample was injected into aSYNAPT™ G2 HDMS™ (Waters Corp.) using Full Scan MS method, with a runtime of 16.5 min total. Peptide(s) of interest were monitored between3-8 min.

Table 5 and FIG. 10 show the average amount of peptide in the CNS samplefrom 5 animals over time after a single dosing of 0.8 mg/kg of thepeptide. FIG. 10 shows the lowest level of quantification at 1.4 μg/mg.

TABLE 5 Single-Dosed Brain PK fmol analyte/ pg/mg Time Point (min) mgtissue of tissue Std. Dev. (pg/mg) 1 10.91 7.79 1.57 3 12.30 8.79 0.67 511.26 8.04 1.51 10 11.83 8.44 1.07 15 10.56 7.54 1.49 30 8.87 6.33 0.8860 7.64 5.46 0.97

Table 6 shows the analyte concentration in 2×3-min brains and 2×10-minbrains. The analyte concentrations were calculated from a single-pointinternal standard quantitation. The preliminary data suggest CNSpenetration. The analyte concentrations were calculated from a 7-pointstandard curve calibration curve equation generated with samples between1.4 μg analyte/mg tissue and 89.2 μg/mg (2× serial dilutions). Theinter-animal reproducibility for the brain analyses is high (greaterthan 21% CV). The drug molecule in the single-dosed brain C_(max) isabout 9 μg analyte/mg tissue.

TABLE 6 Brain Pilot Mouse Time Ratio to fmol/mg pg/mg Sample ID ID Point(min) Heavy (tissue) (tissue) ID07386 11F63 3 0.245 15 11 ID07387 11F643 0.371 23 17 ID07388 11F67 10 0.157 10 7 ID07389 11F68 10 0.435 27 19

Example 9—Effect of ApoE Mimetic Peptide in Murine Stroke Studies

Focal Ischemia-Reperfusion Model

A modified middle cerebral artery occlusion (MCAO) model (Huang et al.,(1994) Science, 265:1883-1885; Laskowitz et al., (1997) J. Cereb. BloodFlow Metab., 17:753-758) was used to determine the effect of the mimeticpeptides on neurological function after stroke and to evaluate thepeptide's efficacy as a therapeutic agent for stroke. The mice wereendotracheally intubated after anesthesia induction with 4.6%isoflurane, and the lungs were mechanically ventilated with 1.6%isoflurane in 30% O₂/70% N₂. Via a midline cervical skin incision, theright common carotid artery was identified. The external carotid arterywas ligated and transected. The internal carotid artery was dissecteddistally until the origin of the pterygopalatine artery was visualized.A 6-0 nylon monofilament with blunted tip lightly coated with siliconewas inserted into the proximal external carotid artery stump andadvanced 11 mm into the internal carotid artery to occlude the middlecerebral artery. After 90 min, the filament was removed to restore bloodperfusion, and the skin incision closed with suture. Isoflurane wasdiscontinued and the mice were extubated upon the recovery ofspontaneous respiration. Post-injury mice were placed in anoxygen-enriched environment (FIO₂=50%) for 1 hr and then returned totheir cages. Rectal temperature was continuously monitored andservoregulated with surface heating/cooling at 37° C. throughout theprocedure.

Testing of Motor Deficits

Two groups of 10-12 week old male C57Bl/6J mice, a control group (n=12)and a VR-55-treated group (n=9), received MCAO. The control group micewere given 100 μL sterile normal saline vehicle by intravenous tail veininjection at 30 min and 6 hrs after reperfusion injury, while theVR-55-treated group mice were given 100 μL sterile normal saline vehicleand VR-55 (SEQ ID NO: 4; 0.05 mg/kg) by intravenous tail vein injectionat 30 min and 6 hrs after reperfusion injury.

An automated Rotorod (Ugo Basile, Comerio, Italy) was used to assessvestibulomotor function, as previously described above. On the daybefore MCAO, the mice underwent 2 consecutive conditioning trials at aset rotational speed (16 rpm) for 60 sec followed by 3 additional trialswith an accelerating rotational speed. The average time to fall from therotating cylinder in the latter 3 trials was recorded as baselinefunctional Rotorod latency. Starting from the first day after MCAO, miceunderwent consecutive daily testing with 3 trials of acceleratingrotational speed (intertrial interval of 15 min) for 3 days. The averagelatency to fall from the rod was recorded. Mice unable to grasp therotating rod were assigned a latency of 0 sec.

As shown in FIGS. 12A and 12B, the control and VR-55-treated mice hadsimilar baseline functional Rotorod latencies. The control mice had abaseline functional Rotorod latency of 217+/−20 sec (“Saline”), whilethe VR-55-treated mice had a baseline functional Rotorod latency of214+/−22 sec (“VR-55”). At Day 1 post-injury, the control andVR-55-treated mice also had similar functional Rotorod latency. However,at Day 3 post-injury, the VR-55-treated mice showed an improvement infunctional Rotorod latency compared to the control mice. TheVR-55-treated mice had a functional Rotorod latency of 216+/−26 secwhile the control mice had a functional Rotorod latency of 161+/−32 sec.These results are consistent with a reduction in delayed neuronal injurysecondary to inflammatory response.

The treated mice are expected to show improved histological endpoints.The other mimetic peptides described herein will be administered invarious dosages and are also expected to show improved functional andhistological endpoints in an MCAO model of mouse stroke.

1. A peptide of Formula I:X1-X2-X3-X4-X5  (SEQ ID NO:1) or a salt thereof, wherein X1 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain; X2 is selected from an aminoacid having a hydrophobic side chain, an amino acid having a positivelycharged side chain, or an amino acid having a polar uncharged sidechain; X3 is selected from an amino acid having a positively chargedside chain; X4 is selected from an amino acid having a positivelycharged side chain; and X5 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain.
 2. The peptide of claim 1, wherein: X1 is V or R; X2 is S, A, orH; X3 is K or R; X4 is K or R; and X5 is R, L, or K.
 3. The peptide ofclaim 2, wherein Formula I comprises VSRKR (SEQ ID NO:2), VSKRR (SEQ IDNO:3), VSRRR (SEQ ID NO:4), VARKL (SEQ ID NO:5), RHKKL (SEQ ID NO:6),RARRL (SEQ ID NO:7), RSKKL (SEQ ID NO:8), RHKRR (SEQ ID NO:9), VARRL(SEQ ID NO:10), VARRK (SEQ ID NO:11), or RSKRR (SEQ ID NO: 12).
 4. Thepeptide of claim 1, wherein the peptide does not have primarypolypeptide sequence identity with any region of 5 consecutive aminoacids of human ApoE protein (SEQ ID NO:14).
 5. The peptide of claim 4,wherein the peptide does not have primary polypeptide sequence identitywith any 5 consecutive amino acids from residue 130 to residue 150 ofhuman ApoE protein (SEQ ID NO: 14).
 6. The peptide of claim 1, whereinthe peptide suppresses activation of microglial cells.
 7. The peptide ofclaim 6, wherein the peptide suppresses secretion of TNF-α by culturedmicroglial cells exposed to lipopolysaccharide.
 8. The peptide of claim6, wherein the peptide suppresses secretion of nitric oxide by culturedmicroglial cells exposed to lipopolysaccharide.
 9. The peptide of claim1, wherein the peptide binds a cell-surface ApoE receptor.
 10. Thepeptide of claim 1, wherein the peptide blocks NMDA receptor mediatedexcitotoxicity.
 11. A method of reducing inflammation in a subject inneed thereof, the method comprising administering to the subject aneffective amount of a peptide of Formula I:X1-X2-X3-X4-X5  (SEQ ID NO:1) or a salt thereof, wherein X1 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain; X2 is selected from an aminoacid having a hydrophobic side chain, an amino acid having a positivelycharged side chain, or an amino acid having a polar uncharged sidechain; X3 is selected from an amino acid having a positively chargedside chain; X4 is selected from an amino acid having a positivelycharged side chain; and X5 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain.
 12. The method of claim 11, wherein the method comprisingadministering the peptide by an injection, inhalation, transdermal,intravenous, intranasal, intracranial, and/or intrathecal route.
 13. Amethod of treating a neurological condition in a subject in needthereof, the method comprising administering to the subject acomposition comprising an effective amount of a peptide of Formula I:X1-X2-X3-X4-X5  (SEQ ID NO:1) or a salt thereof, wherein X1 is selectedfrom an amino acid having a hydrophobic side chain or an amino acidhaving a positively charged side chain; X2 is selected from an aminoacid having a hydrophobic side chain, an amino acid having a positivelycharged side chain, or an amino acid having a polar uncharged sidechain; X3 is selected from an amino acid having a positively chargedside chain; X4 is selected from an amino acid having a positivelycharged side chain; and X5 is selected from an amino acid having ahydrophobic side chain or an amino acid having a positively charged sidechain.
 14. The method of claim 13, wherein the neurological condition isselected from at least one of traumatic CNS injury, subarachnoidhemorrhage, intracranial hemorrhage, stroke, experimental allergicencephalomyelitis, multiple sclerosis, neuroinflammation, chronicneurological disease, ALS, dementia, neuropathy, epilepsy, Parkinson'sdisease, and Alzheimer's disease.
 15. A peptide of claim 1, wherein saidpeptide is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 16. A method of claim 11,wherein said peptide is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 17. A method of claim 13,wherein said peptide is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.